Advanced Glycation End-products (AGEs) are adducts that form on proteins incubated in the presence of sugar. As the name implies, it has been suggested that accumulation of AGEs on our long-lived tissue proteins may play a role in human aging and its associated diseases.
Advanced Glycation End-product modified Bovine Serum Albumine (AGE-BSA) is a simple model for studying the biochemistry of AGEs, and their effects on cultured cells and tissues. Our AGE-BSA has been used in drug screening assays aimed to mitigate the adverse effects of AGEs.
When working with AGE-BSA, there are numerous pitfalls. BSA is a carrier protein for a great variety of hydrophobic substances in mammalian blood, including lipids, hormones, vitamins and pharmaceuticals. Each batch of commercially available BSA may have different substances attached to it. How can you be sure you are measuring the effects of AGEs, and not some other difference in the many substances that could be bound to your AGE-BSA and your control BSA? And what happens to these attached substances and the BSA itself during the long incubation needed to produce AGE-BSA? What are the best practices to make sure you are measuring the effects of AGEs and not some poorly understood other difference? Few literature papers, and no commercial vendors we're aware of even attempt to address these questions.
But now you can. We are giving you not just AGE-BSA, but a set of controls from the same batch, including fresh-BSA and control-incubated BSA. Furthermore, we eliminate most unwanted substances by HPLC-purification before and after each incubation. We do not use impure serum "fractions" widely sold as BSA.
AGE-BSA Preparation Details
AGE-BSA and controls are manufactured at our facility in California. A large batch of "BSA fraction V" from a major US vendor is the starting material. BSA is purified by Size-Exclusion HPLC. The BSA-containing eluates are pooled, mixed and then divided in three equal lots. An appropriate amount of ACS-grade glucose is added to the lot destined to become AGE-BSA. All three fractions are sterilized by filtration through 0.22 uM PES. AGE-BSA and control BSA are incubated at 50C for 8 weeks and inspected weekly. Any amount of visible precipitation disqualifies a batch. Fresh BSA is frozen to -80C for the entire time.
After the 8-week incubation, each lot is again purified by Size-Exclusion HPLC. They are then filter-sterilized, filled into sterile vials under aseptic conditions and lyophilized.
AGE-BSA Product Characteristics
AGE-BSA undergoes profound spectral changes, turning dark brown over the 8 weeks. This is evident in the absorbance spectra of the three products (equal amounts of each are analyzed):
AGE-BSA has undergone dramatic spectral changes at all UV-vis wavelengths. Especially pronounced is the gain in the visible spectrum >350 nm, where both control and fresh BSA remain transparent. The specific ratio of A280 / A370 can be used to gauge the degree of AGE formation, compare products from different vendors etc. Ours is 0.256 for the batch shown here, and is guaranteed > 0.230.
AGEs are not just absorbing light, but can also emit it back as fluorescence. The fluorescence spectra of AGE-BSA and controls are shown below. The typical excitation wavelength of 370 nm was used here:
It is evident that not just AGE-BSA, but also control-incubated BSA has gained some fluorescence! This is not due to glycation. We removed all glucose from the starting material by Size Exclusion HPLC, and verified its absence experimentally. Also, control-incubated BSA showed no browning at all (see absorbance spectrum above). Therefore, the gain in fluorescence is most likely due to some other change that affected control BSA during its long incubation period. We have not characterized what it is, but oxidation seems like a likely culprit.
This gain in fluorescence in the absence of glycation is one small example for how the two controls, fresh and control-incubated BSA, can be used to tease out whether a given change is due to glycation specifically. Whatever effect of AGE-BSA you are measuring, be it biochemically or on living cells, with our set of highly purified same-batch controls, you will always be ideally positioned to assess to what degree any phenomenon is caused specifically by AGEs, versus some other process that also affected the controls.
Bacterial endotoxin is a potentially critical contaminant in many AGE-BSA experiments, because it activates similar pro-inflammatory pathways as AGEs do. Therefore, we test it directly for each batch, and guarantee it to be orders of magnitude below where it would start to interfere in cell-based assays. Typical results are shown below.
We dissolved the entire contents of a 20 mg vial in 2 ml endotoxin-free water. We prepared dilutions as shown in the table below, to push the signal into the middle of the measurable range. We then measured endotoxin using a colorimetric kit based on the limulus amoebocyte lysate method from a major US vendor:
As shown in the table, we express the results as Endotoxin Units per milligram of protein, by back-calculating our resuspension volume and any dilutions made. We guarantee AGE-BSA and controls to contain below 1 EU per mg of protein, and in practice typically score much lower. This is all far below the concentration range where endotoxin would be a concern. We encourage you to do the math based on your own experimental aims -- but rest assured that this will be orders of magnitude below where you would see endotoxin effects, even if you use ungodly amounts of AGE-BSA.
Gene And Cell Technologies AGE-BSA and controls are lyophilized powders. They ship overnight at room temperature, and are stable in this state for weeks. Upon arrival, they should be stored in the fridge for the short term, and at -80C or -20C for the long term.